Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Wine is a complex medium. Wine aroma, flavour and colour are important quality
factors, but these can be influenced by many factors, such as grape-derived
compounds that exist as free volatiles and also as glycosidically bound. The
chemical composition of wine is determined by factors such as grape variety,
geographic position, viticulture condition, microbial ecology of the grape and the
winemaking process. The varietals aroma is determined by both the volatile and
the non-volatile compounds, such as monoterpenes, norisoprenoids and benzene
derivatives, which are naturally present in the wine. Monoterpenes are very
important in the flavour and aroma of grapes and wine. They can be found in
grapes and wine either in the free, volatile and odorous form, or in the
glycosidically-bound, non-volatile and non-odorous form. The ratio of
glycosidically-bound compounds to free aroma compounds is very high in the
Gewürztraminer, Muscat and Riesling cultivars in particular.
The glycosidic bonds can be hydrolysed either by the acid method or by using
enzymes. The acid method is disadvantageous because it can modify the
monoterpenes, whereas enzymatic hydrolysis has the advantage of not modifying
the aroma character. The enzyme method of breaking the glycosidic bonds occurs
in two successive steps: initial separation of glucose from the terminal sugar by a
hydrolase (a-L-arabinofuranosidase, a-L-rhamnosidase or β-apiosidase,
depending on the aglycone moiety), followed by the breaking of the bond between
the aglycone and glucose by β-glucosidase.
The enzyme β-glucosidase can be obtained from many plant (Vitis vinifera),
bacterial, yeast or fungal sources. Most of the enzymes produced by these sources
are not functional under the winemaking conditions of low pH, low temperature,
high glucose and high ethanol content. However, β-glucosidases from fungal
origins, particularly from Aspergillus spp., are tolerant of winemaking conditions.
The idea of using the β-glucosidase gene from the fungus Aspergillus kawachii
(BGLA), which is linked to the cell wall and the free β-glucosidase, was to
determine if anchoring the enzyme to the cell wall will increase the activity of the
enzyme compared to the free enzyme. Four plasmids, pCEL 16, pCEL 24, pDLG
97 and pDLG 98, were used in this study. BGLA that was cloned into the plasmids
pCEL 24 and pDLG 97 was linked to CWP2, and in pDLG 98 it was linked to AGa1
anchor domains. All the plasmids were genome-integrated and expressed in the
reference strain Saccharomyces cerevisiae 303-1A. All the transformants were
grown in 2% cellobiose and showed higher biomass production compared to the
reference strain. β-Glucosidase activity was also assayed and transformed strain
W16 showed a fourfold increase in activity compared to the reference strain. There
was no significant increase in the activity of the other transformed strains, W24,
W97 and W98. Enzymatic characterisation for optimum pH and temperature was done – for all strains the optimum pH was 4 and the optimum temperature was
40ºC.
The recombinant strains together with the reference strain were used to make
wine from Gewürztraminer grapes. The levels of numerous monoterpenes were
enhanced in the resultant wines. The concentration of nerol was increased
fourfold, that of citronellol twofold, and geraniol was 20% higher than in the wild
type. There was also an increase in the levels of linalool and a-terpinol, but this
was not significant. In wines produced with W97, W98 and W24, monoterpene
levels did not show a significant difference.
In future, the expression of the W16 expression cassette in an industrial wine
yeast strain could be performed. In combination with the production of enzymes
such as a-arabinofuranosidase, a-rhamnosidase and β-apiosidase, which are
involved in the first step of enzymatic hydrolysis, this wine strain could release the
bound monoterpenes and enhance the aroma of the wine. / AFRIKAANSE OPSOMMING: Wyn is ‘n komplekse medium. Wynaroma, -geur en -kleur is belangrike
kwaliteitsfaktore, hoewel hierdie kwaliteite deur verskeie faktore beïnvloed kan
word, soos druifafgeleide verbindings wat as vry vlugtige stowwe teenwoordig kan
wees of glikosidies gebind is. Die chemiese samestelling van wyn word bepaal
deur faktore soos druifvariëteit, geografiese ligging, wingerdkundige toestande,
mikrobiese ekologie van die druif en die wynbereidingsproses. Die variëteitsaroma
word bepaal deur vlugtige en nie-vlugtige verbindings, soos monoterpene,
norisoprenoïede en benseenderivate, wat natuurlik in die wyn voorkom.
Monoterpene is baie belangrik vir die geur en aroma van druiwe en wyn.
Monoterpene is teenwoordig in die druiwe en wyn in vry, vlugtige en geurige, of in
glikosidiesgebinde, nie-vlugtige en nie-geurige vorms. Die verhouding van
glikosidiesgebonde verbindings tot vry aromaverbindings is baie hoog, veral in die
Gewürztraminer-, Muscat- en Riesling-kultivars.
Glikosidiese verbindings kan deur óf die suurmetode óf die ensiemmetode
gehidroliseer word. Die nadeel van die suurmetode is dat dit monoterpene kan
modifiseer, terwyl die ensiemmetode die voordeel het dat dit nie die aromakarakter
modifiseer nie. Die ensiemmetode waarmee die glikosidiese verbinding afgebreek
word, vind in twee opeenvolgende stappe plaas: aanvanklike skeiding van glukose
van die terminale suiker deur ‘n hidrolase (a-L-arabinofuranosidase, a-Lramnosidase
of β-apiosidase, afhangende van die aglikoongedeelte), gevolg deur
die verbreking van die verbinding tussen die aglikoon en glukose deur β-
glukosidase.
Die β-glukosidase-ensiem kan vanaf ‘n verskeidenheid plant- (Vitis vinifera),
bakterie-, gis- en swambronne verkry word. Die meerderheid van die ensieme wat
deur hierdie bronne geproduseer word, is nie onder die wynbereidingstoestande
van lae pH, hoë temperatuur, hoë glukose en hoë etanol funksioneel nie. β-
Glukosidase vanaf ‘n swamoorsprong, veral vanaf Aspergillus-spesies, kan egter
wynbereidingstoestande verdra.
Die idee agter die gebruik van die β-glukosidasegeen afkomstig van die swam
Aspergillus kawachii (BGLA), wat aan die selwand en die vry β-glukosidase
gekoppel is, was om te bepaal of die aktiwiteit van die ensiem in vergelyking met
dié van die vry ensiem verhoog sou word indien die ensiem aan die selwand
geanker is. Vier plasmiede, pCEL 16, pCEL 24, pDLG 97 en pDLG 98, is in hierdie
studie gebruik. BGLA, wat in die plasmiede pCEL 24 en pDLG 97 gekloneer is, is
gekoppel aan CWP2, en in pDLG 98 is dit aan AGa1-ankergebiede gekoppel. Al
die plasmiede is in verwysingsras Saccharomyces cerevisiae 303-1A
genoomgeïntegreer en uitgedruk. Al die transformante is in 2% sellobiose gegroei
en het hoër biomassaproduksie as die verwysingsras getoon. β-Glukosidaseaktiwiteit
is ook geëssaieer en die getransformeerde ras W16 het ‘n viervoudige verhoging in aktiwiteit in vergelyking met die verwysingsras getoon. Daar was geen
noemenswaardige verhoging in die aktiwiteit van die ander getransformeerde
rasse, W24, W97 en W98, nie. Ensimatiese karakterisering vir optimum-pH en -
temperatuur is gedoen – vir al die rasse was die optimum-pH 4 en die
optimumtemperatuur 40ºC.
Die rekombinante rasse, tesame met die verwysingsras, is gebruik om wyn
met Gewürtztraminer-druiwe te maak. Die vlakke van talryke monoterpene is in die
gevolglike wyne verhoog. Die konsentrasie van nerol is viervoudig verhoog, dié
van sitronellol tweevoudig, en geraniol was 20% hoër as in die wilde tipe. Daar
was ook ‘n verhoging in die vlakke van linaloöl en a-terpinol, maar hierdie
verhoging was nie noemenswaardig nie. In wyne wat met W97, W98 en W24
gemaak is, het die monoterpeenvlakke nie ‘n noemenswaardige verskil getoon nie.
In die toekoms sal die uitdrukking van die W16-uitdrukkingskasset in ‘n
industriële wyngisras uitgevoer kan word. In kombinasie met die produksie van
ensieme soos a-arabinofuranosidase, a-ramnosidase, β-apiosidase, wat in die
eerste stap van ensimatiese hidrolise betrokke is, sal hierdie wyngisras die
gebonde monoterpene kan vrylaat en die aroma van die wyn kan verbeter.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21731 |
| Date | 12 1900 |
| Creators | Ranwedzi, Ntanganedzeni |
| Contributors | Van Rensburg, P., Cordero Otero, Ricardo R., Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 44 leaves : ill. |
| Rights | Stellenbosch University |
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